The present invention relates generally to an immunologically reactive molecule (IRM) capable of binding to a target molecule from at least one species of insect but not from at least one other species of insect. The present invention also contemplates a method of distinguishing between, or identifying, one or more insect species or subspecies.
Insect damage to crops is a significant factor in decreasing crop yield and considerable effort is expended annually in controlling insect populations to minimise or prevent crop damage. Although chemical insecticides have played an important role in controlling insect populations, increasing resistance to these insecticides and environmental concerns have rendered the use of chemical insecticides less appealing.
Resistance Management Strategies (RMS""s) have been developed in order to minimize insecticide resistance and usually involve the administration of more than one insecticide. However, such strategies are significantly more expensive, are time consuming and do not address the environmental concerns.
The insect genera Heliothis and Helicoverpa comprise economically important pests which attack a range of crops including cotton, tobacco, maize, sorghum, sunflower, soybean, chick peas and other pulses, rapeseed, canola, ground nuts, lucerne and many horticultural crops such as cut flowers and tomatoes and other vegetables (Fitt, 1989).
Helicoverpa (Heliothis) armigera and Helicoverpa (Heliothis) punctigera (Lepidoptera: Noctuidae) (hereinafter referred to as H. armigera and H. punctigera) cause crop damage which is economically significant to agricultural industry. Whilst H. punctigera is the more abundant species in Australia, H. armigera is considered the more problematic of the two because it has a history of developing resistance to the chemical insecticides which have been used in its control (Fitt, 1989). Helicoverpa punctigera, however, does not appear to have the same propensity for developing resistance to insecticides.
Heliothis virescens and Helicoverpa zea (hereinafter referred to as H. zea) are important pests of crops throughout North America and especially crops in the U.S.A. Whilst both species have developed resistance to some insecticides, (Fitt, 1989) they are distinguished by the fact that Heliothis virescens may display up to 150 fold resistance to synthetic pyrethroids whereas H. zea shows no detectable resistance to these compounds (Elzen et al., 1992). Synthetic pyrethroids are therefore more efficacious for control of H. zea than for Heliothis virescens. 
Heliothis virescens is congeneric with Heliothis (Neocleptria) punctifera whereas H. zea is congeneric with Heliocoverpa armigera and Helicoverpa punctigera. 
Resistance Management Strategies developed for H. armigera and H. punctigera have involved a rotation of pyrethroids, endosulfan and other insecticides to reduce exposure to endosulfan and pyrethroids. This has been necessary due to the increasing resistance to synthetic pyrethroids and endosulfan. Despite these RMS""s, the frequency of resistant individuals has been steadily increasing (Forrester, 1990).
Helicoverpa armigera and H. punctigera at their egg and neonate stages are morphologically indistinguishable, whilst their respective larvae can only be distinguished by experts. A similar situation exists for Heliothis virescens and H. zea. Current RMS therefore make assumptions based on historical records of the relative abundance of the two species, averaged over wide geographical distances and several seasons. These assumptions necessarily entail some degree of inaccuracy, the consequence of which is that, in some cases, expensive alternative insecticides may be applied to H. punctigera, whilst in other cases, there may be multiple applications of pyrethroids to populations of H. armigera. In the latter case, the pyrethroid applications may be ineffective and will generally encourage the emergence of insecticide resistance. Similar problems also arise with Heliothis virescens and H. zea. Such situations may lead to unnecessarily expensive insect control for farmers. For example, insecticides such as pyrethroids which are adequate for species without a propensity to develop resistance are comparatively cheap insecticides. These may cost the farmer 3 or 4 times less to apply than those insecticides which are applied to insect species with a tendency to develop insecticide resistance. There is a need, therefore, to develop a convenient method to determine the species composition of egg lays, neonates or older larva which may have survived an insecticide application in the field. Such a method would be particularly useful for routine use by crop growers/farmers, agronomists and/or agricultural scientists so that insecticide applications can then be tailored to the composition of the Heliothis or Helicoverpa population actually present.
In work leading up to the present invention, the present inventors have found an immunologically reactive molecule (IRM) that distinguishes between insect species or subspecies. The IRM is capable of binding to a target molecule in at least one species or subspecies but does not react with at least one other species or subspecies. This provides a basis for distinguishing between or identifying insect species or subspecies.
Accordingly, one aspect of the invention relates to an immunologically reactive molecule (IRM) capable of binding to a target molecule from at least one species or subspecies of insect but not to at least one other species or subspecies of insect. The target molecule is an antigen which is present in insect tissues. These tissues may be those of eggs, neonates or older larvae.
Another aspect of the present invention contemplates a method of identifying one or more species or subspecies of insect by determining the reactivity of an IRM to a target molecule in a sample of insect material, wherein said IRM is capable of binding to the target molecule present in at least one of said species or subspecies but substantially not to any molecule of at least one other species or subspecies of insect. More particularly, the method comprises contacting a sample of insect material with an effective amount of an IRM specific for said target molecule of the one or more species or subspecies of insect for a time and under conditions sufficient for a complex to form between the target molecule and IRM, and then subjecting said complex to a detecting means.
Another aspect of the present invention relates to a kit for distinguishing between or identifying one or more insect species or subspecies from at least one other insect species or subspecies, said kit comprising in compartmentalised form a first compartment adapted to receive an IRM capable of binding to a target molecule of at least one species or subspecies of insect but not at least one other species or subspecies of insect and at least one other compartment adapted to contain a detector means.
In another aspect the instant invention relates to an isolated or purified preparation of a target molecule as herein described specific for a particular insect species or subspecies, to mutants and derivatives thereof and to the antigenic determinants of the target molecule.
In another aspect of the invention there is provided a method for making antibodies, particularly monoclonal antibodies, to said antigenic determinants for use in a method of detecting particular insect species. Antibodies can be made by standard techniques such as immunizing animals with insect extracts from said at least one species or subspecies and collecting the serum, identifying antibodies from said serum which are capable of binding at least one species or subspecies but not at least one other species or subspecies (Campbell (1984), Goding (1983) and Harlow and Lane (1988)).
The present invention is predicated in part on the surprising discovery that different species or subspecies of insects that are morphologically indistinguishable for at least a part of their life cycles can be differentiated by an IRM which is capable of binding a target molecule present in one species or subspecies but is not capable of binding a molecule in one other species or subspecies.
Accordingly, one aspect of the invention relates to an IRM capable of binding to a target molecule from at least one species or subspecies of insect but not to at least one other species or subspecies of insect. The target molecule is an antigen which is present in insect tissues. These tissues may be those of eggs, neonates or older larvae. Preferably the target molecule is present in older larvae and neonates, more preferably the target molecule is present in eggs, still more preferably it is present in eggs and neonates. Even more preferably the target molecule is present in eggs, neonates and older larvae. Most preferably the target molecule is abundantly present in all life stages of the insect, including the egg, neonate and older larval stages. In one aspect of the invention the target molecule is lipophorin, a protein which is present in insects at all stages during their life cycles.
The term xe2x80x9csubspeciesxe2x80x9d used herein refers to different strains, varieties, serotypes, subserotypes and/or other categories/ groups/strains/varieties of insects within one species, genus or order. Although specific Lepidoptera are mentioned herein the invention covers Lepidoptera generally and other insect groups.
Preferably the IRM is in isolated form meaning it is in a state different to that found in nature. Advantageously, the isolated IRM is biologically pure meaning that a composition comprises, at least 20%, preferably at least 30% more preferably at least 40-50%, still more preferably at least 60-75%, and more preferably at least 85-95% of IRM as determined by weight, activity, immunological reactivity or other convenient means.
The IRM is preferably an antibody and advantageously a monoclonal antibody (mAb). The term xe2x80x9cantibodyxe2x80x9d includes naturally occurring antibodies, recombinant antibodies, synthetic antibodies including fusions or chimers of antibodies and fragments of any of the foregoing such as Fab and F(abxe2x80x2)2. Where the antibody IRM is a recombinant form, the molecule may be encoded by a naturally occurring or synthetic nucleotide sequence and expressed in any convenient expression system. Where the molecule is synthetic, it is conveniently prepared by the step-wise addition of single amino acid groups or amino acid fragments of, for example antibodies. With regard to the latter, the synthetic antibody may be a fusion or chimeric antibody comprising light or heavy chains derived from other antibodies.
Antibodies and other IRM""s of the present invention may be of any animal origin including mammals such as humans, live stock animals, companion animals, wild animals and laboratory test animals (eg. mice, rats, rabbits and guinea pigs). An xe2x80x9canimalxe2x80x9d also extends to non-mammalian species such as birds (eg. chickens and other poultry, emus and ostriches).
The IRM may be labelled with a reporter providing, under suitable conditions, a detectable signal. Such reporters may include molecules such as radio-nucleotides, chemiluminescent molecules, bioluminescent molecules, fluorescent molecules or enzymes amongst others. Commonly used enzymes include horseradish peroxidase, glucose oxidase, xcex2-galactosidase and alkaline phosphatase, amongst others. Alternatively the reporter may consist of particles including colloidal gold, latex microbeads or erythrocytes amongst others. In a further alternative the reporter may comprise a physical detector means such as biosensors, for example surface plasmon resonance.
Although not intending to limit the present invention to any one theory or proposed mode of action, the basis of the differential reactivity of the immunologically reactive molecules may be due to differences in antigenic determinants on the molecule for which they are specific. This could be due to changes or alterations in protein, carbohydrate or lipid molecules or combinations thereof, or to conformational changes, whether or not resulting therefrom. Thus in the case of lipophorin, in one embodiment of the invention, the IRM is reactive with the lipophorin of H. armigera but not the lipophorin of H. punctigera. 
In a preferred aspect of the present invention the IRM is capable of binding a target molecule of at least one species or subspecies of Helicoverpa or Heliothis but not at least one other species or subspecies of Helicoverpa or Heliothis. Preferably the IRM is an antibody capable of binding to a target molecule of H. armigera not H. punctigera. Alternatively, preferably the IRM is capable of binding a target molecule of H.zea but not Heliothis virescens. Preferably, the target molecule for which the IRM is specific is an antigen present in egg or neonate tissues.
In its most preferred form, the present invention provides monoclonal antibodies mAb 70.5 and mAb 70.7 which bind H. armigera not H. punctigera. A hybridoma producing mAb 70.5 was deposited on Jan. 21, 1993 at the European Collection of Animal Cell Cultures, Public Health Laboratory Service, Porton Down, Salisbury, U.K. and given accession number 93012101.
In another most preferred form, the present invention provides monoclonal antibody mAb 21.91.2 which binds H. zea, H. armigera, or H. punctigera and not to Heliothis virescens or Heliothis (Neocleptria) punctifera. 
Another aspect of the present invention contemplates a method of identifying one or more species or subspecies of insect by determining the reactivity of an IRM to a target molecule which may be present in a sample of insect material, wherein said IRM is capable of binding to a target molecule present in at least one of said species or subspecies but substantially not to any molecule of at least one other species or subspecies. More particularly, the method comprises contacting a sample of insect material with an effective amount of an IRM specific to said target molecule of the one or more species or subspecies of insect for a time and under conditions sufficient for a complex to form between the target molecule and IRM, and then subjecting said complex to a detecting means.
The term xe2x80x9ca sample of insect materialxe2x80x9d used herein means a sample of insect dervied from any life stage, or part of an insect, such as the eggs, larval or adult stages.
Preferably, IRM is an antibody as defined above, and is most preferably a mAb. Most preferably, the antibody is specific for a target molecule of H. armigera not H. punctigera. Alternatively most preferably the antibody is specific for a target molecule of H. zea not Heliothis virescens. Still more preferably the antibody is specific for an antigen present in egg or neonate tissues. Most preferably the IRM is specific for lipophorin of H. armigera not H. punctigera. Suitable antibodies include mAb 70.5 and 70.7. Alternatively the IRM is specific for a target molecule present in H. zea not H. virescens. Suitable antibodies include mAb 21.91.2.
Detection of binding of the IRM to the target molecule can be determined by any convenient means. Preferably, the target molecule is detected by an IRM which is labelled directly by a reporter as herein described, such as by a reporter enzyme, or by reporter particles such as colloidal gold or by physical means such as biosensors. Alternatively, the presence of antigen-antibody complex may be detected by an anti-immunoglobulin labelled with a label, reporter or other detector molecule capable of providing a detectable signal. In this embodiment, the anti-immunoglobulin binds to the bound target molecule reactive antibody and then the label, reporter or detector molecule is read. Suitable reporters are as hereinbefore described.
The target molecule containing sample is preferably in the form of eggs or larvae but may be from later life stages of the insect. The sample may be contacted with, preferably squashed onto any conventional material such as a polymer or glass, the most commonly used polymers being cellulose (including paper) nitrocellulose, polyacrylamide, nylon, polystyrene, polyvinylchloride, polyvinylidene difluoride (PVDF) or polypropylene.
The immunologically reactive molecule of the present invention may also be bound to a solid support such as those listed above.
Another aspect of the present invention relates to a kit for distinguishing between, or identifying, one or more insect species or subspecies from at least one other insect species or subspecies, said kit comprising in compartmentalised form a first compartment adapted to receive an IRM capable of binding to a target molecule of at least one species or subspecies of insect but substantially not at least one other species or subspecies of insect, and at least one other compartment adapted to contain a detector means. In a preferred form, the IRM is bound to a support as herein before described, more preferably a solid support. Most preferably the solid support comprises sheet material such as a membrane.
Preferably the kit comprises an IRM reactive with H. armigera not H. punctigera. Most preferably the kit comprises mAb 70.5 or mAb 70.7.
The detection means preferably comprises a reporter or label as described herein which can detect the binding of the IRM to the target molecule.
In one preferred aspect, the kit comprises a mAb specific for the target molecule directly conjugated with a detectable reporter such as an enzyme, and where appropriate enzyme substrate and suitable buffers. In a more preferred aspect the kit comprises mAb 70.5 or 70.7 conjugated with horseradish peroxidase, alkaline phosphatase or xcex2-galactosidase, enzyme substrate and appropriate buffers.
In another preferred aspect the kit comprises an anti-immunoglobulin which binds the antibody-target molecule complex. The anti-immunoglobulin is labelled with a label, reporter or other detector as herein described capable of providing a detectable signal.
In another aspect the instant invention relates to an isolated or purified preparation of a target molecule as herein described specific for a particular insect species or subspecies, to mutants and derivatives thereof and to the antigenic determinants of the target molecule. The antigenic determinants of the instant invention may comprise the amino acid/polypeptide, lipid and/or polysaccharide portion of the target molecule. Such mutants and derivatives of the target molecule or the antigenic determinants thereof may be produced by natural chemical and/or recombinant means.
The term xe2x80x9cmutantxe2x80x9d used herein refers to a variant of apolipophorin which while different from native apolipophorin retains substantially the same biological or immunological characteristics of apolipophorin. The term xe2x80x9cderivativexe2x80x9d used herein refers to a molecule derived from apolipophorin which while different from native apolipophorin retains substantially the same biological or immunological characteristics as apolipophorin.
In a most preferred embodiment the invention relates to a purified preparation of lipophorin, the 70 kDa apolipophorin II and 230 kDa apolipophorin I subunits of lipophorin from H. armigera, H. punctigera, H. zea or Heliothis virescens and to mutants and derivatives thereof.
In another aspect of the invention there is provided a method for making antibodies, particularly monoclonal antibodies, to said antigenic determinants for use in a method of detecting particular insect species. Antibodies can be made by standard techniques such as immunizing animals with insect extracts from said at least one species or subspecies and collecting the serum, identifying antibodies from said serum which are capable of binding at least one species or subspecies but not at least one other species or subspecies (Campbell (1984), Goding (1983) and Harlow and Lane (1988)). Alternatively the antibodies can be made by isolating immune cells from animals which have been immunised with insect extracts and fusing the immune cells with immortalising cells to produce immortalised cells and identifying antibodies from the immortalised cells which are capable of binding to a molecule from at least one species or subspecies of insect but not to at least one other species or subspecies of insect. Similarly monoclonal antibodies can be made by in vitro immunisation which involves taking immune cells from an animal, which animal may or may have been immunised with an insect extract, exposing the cells so removed to the insect extract and fusing the cells with an immortalising cell line as described above. The immortalising cells may be screened in the manner described above. As stated above mAbs can be made according to standard techniques including that described below.
The present invention is further described by reference to the following non-limiting figures at Example.